Excitatory synaptic transmission in the mammalian brain is primarily mediated by the neurotransmitter glutamate acting on two classes of postsynaptic glutamate receptors: NMDA receptors and AMPA receptors (NMDARs and AMPARs). Our long-term objective is to understand the molecular mechanisms by which glutamate receptors are targeted to the postsynaptic membrane and how these mechanisms can be regulated to control the strength of synaptic transmission. This proposal therefore addresses a fundamental issue in synaptic plasticity, which is of relevance to brain development and to learning and memory. Recent studies suggest that regulated trafficking of postsynaptic AMPARs is a major mechanism underlying the long-lasting potentiation and depression of synaptic transmission. However, the cell biological events have only been superficially (and largely qualitatively) described, and the underlying molecular mechanisms remain poorly understood. To address these important questions, we will use quantitative immunocytochemical and biochemical techniques to comprehensively investigate AMPAR internalization (endocytosis) and insertion (exocytosis) in cultured hippocampal neurons. The molecular mechanisms by which AMPAR internalization/insertion is regulated by synaptic activity and growth factors will be studied with subunits GluR1 and GluR2. The site of new AMPAR insertion in the neuronal surface will be visualized at high resolution. In addition, we will search for and functionally characterize novel AMPAR-binding proteins that may play a role in AMPAR trafficking, and hence synaptic plasticity. Finally, the functional significance of the molecular findings will be tested by electrophysiological experiments in hippocampal slices. In each of these aims, we have strong Preliminary Data that substantiate the significance and that demonstrate the feasibility of the proposed experiments. Because glutamate receptor function is intimately involved in brain synaptic function and excitotoxicity, this research may be relevant to human acute and chronic neurodegenerative disease and to psychiatric disorders.